In recent years, avian influenza outbreaks have occurred worldwide and affect humans to cause severe disease conditions, thereby attracting much attention. In particular, the spread of avian influenza in the poultry industry causes devastating economic losses. Accordingly, there is an urgent need to develop a vaccine to protect birds against avian influenza.
The avian influenza virus is an RNA virus belonging to the family Orthomyxoviridae, of which the genome is split to 8 RNA segments and easily modified by recombination. The influenza virus is characterized by having multiple serotypes, and no cross protection occurs between different serotypes. Currently, the virus is largely classified into three types (A, B, and C) according to antigenic differences in the nucleoprotein and the matrix protein, and all of the animal influenza viruses belong to type A.
The influenza virus type A has two proteins, hemagglutinin (HA) and neuraminidase (NA) on its surface. Hemagglutinin (HA) has a binding site with sialic acid that is present in host bronchial epithelial cells, and enters the nucleus via receptors to produce new influenza virus particles. Neuraminidase (NA) cleaves the linkage between sialic acid and viral HA to destroy the receptors, resulting in preventing the accumulation of newly produced viral particles on the infected cell membrane.
Hemagglutinin (HA) is classified into 16 HA subtypes (H1˜H16), and neuraminidase (NA) into 9 NA subtypes (N1˜N9) according to the antigenic properties, which potentially allows 144 various combinations of the influenza virus type A. Among them, the subtypes H5 and H7 are known to be pathogenic to birds, and the subtypes H1, H2, and H3 are known to cause influenza in humans. It has been known that avian influenza virus does not infect any animal, other than avian and swine species. However, an H5N1 avian influenza virus was isolated from patients in Hong Kong in 1997, which confirmed the possibility of human infection by the avian influenza virus. The human infection is thought to be caused by highly pathogenic viruses, which are generated by genetic mutation of avian influenza virus and human influenza virus, when they simultaneously infect human. These highly pathogenic viruses may transmit from person to person, and the risk of pandemic is increasing. Therefore, there is a need for developing a safe vaccine for birds, which is a host for avian influenza virus.
However, the avian influenza virus has a variety of serotypes, and there is little cross-immunity among the subtypes. Thus, it is hard to prevent the infection by other serotypes. Further, since the avian influenza viruses are highly apt to undergo mutation, there is no effective vaccine for preventing the avian influenza. Currently, the most effective prevention method is washing with antiseptic agents, and parenteral vaccination with inactivated influenza virus vaccine or a recombinant fowl pox virus vaccine. However, such methods can be performed, even after being infected with avian influenza and examination of the subtype of virus. Further, these vaccines may reduce the amount of virus in feces from infected poultry, but hardly prevent the spread of the disease and also incur high cost.
As alternatives, oral vaccine and edible vaccine that are directly applied to mucous membranes are suggested, and many studies have been conducted thereon, for example, oral administration of avian influenza virus itself (John M. Crowford et al., Avian disease, (1998), 42(3):486-96)) and oral spray immunization with influenza virus. However, for the preparation of these vaccines, attenuated virus or viral proteins expressed in bacteria are used, which are different from those expressed in mammals.
Meanwhile, a plant has a eukaryotic protein synthesis pathway, in which post-translational modifications being essential for mammals occur (Cabanes-Macheteau et al., Glycobiology 9.365-372 (1999)). Thus, plants are able to produce proteins similar to those expressed in mammals. For that reason, much of the focus has been placed on the production of desired proteins using transgenic plants.
The present inventors have expressed an HA or NA protein specific to avian influenza virus in transgenic plants using their properties, and established the condition for mass-production to develop an avian influenza vaccine of plant origin, which is a safe and economical mucosal vaccine, thereby completing the present invention.